This invention relates to the field of agglutination assays to detect the binding of ligands, and particularly immunological binding (antigen and antibody binding) such as that involved in blood group serology (“immunohematology”).
Immunohematology requires the determination of blood cell compatibility between a blood donor and a patient recipient before a transfusion or organ transplant involving the patient. Blood cell compatibility is determined by the non-occurrence of an immunological reaction between antibodies contained in the blood serum of a patient and antigens present on blood cells from the donor.
Many different blood group antigens are found on the surface of the red blood cells of every individual. These antigens, the products of inherited genes, exist in a unique combination in everyone except identical twins. Blood grouping is generally the process of testing red cells to determine which antigens are present and which are absent, normally utilizing antibodies to the antigen tested for.
Additionally, when a person does not have a particular red cell antigen on his or her red blood cells, his or her serum may contain antibody to that antigen. Whether or not the antibody is present in the serum depends on whether the person's immune system has been previously challenged by, and responded to, that specific antigen or something very similar to it. For example, a person whose red blood cells are Type A, i.e. having “A” antigens on the red cells, will have anti-B antibodies in his or her serum. Thus, if such a person is given type B blood, an immunological reaction will occur with possible serious clinical consequences.
As an additional consideration, it should be noted that the human body is constantly exposed to antigens in pollens, food, bacteria and viruses. Some of these “natural” antigens are apparently so similar to human blood group antigens that they stimulate almost every susceptible person to produce antibodies. Thus, certain antibodies are expected in the serum of anyone whose red cells lack the reciprocal antigen. This is especially true of the ABO system. Accordingly, a second confirmatory test is often performed on the patient/donor sera. The test for expected antibodies of the ABO blood group system in sera is called “reverse” or “indirect” blood grouping, whereas the test for antigen on patient red blood cells (“RBCs”) is referred to as “forward” or “direct” testing.
Since the early 1900's, the general approach, known as the “Landsteiner” method, has been to add a patient's red blood cells to a standard laboratory test tube containing a blood group antibody (such as Anti-A or Anti-B) mix to allow antibody/antigen binding reactions to take place, and then to centrifuge. If the antigen tested for is present, the antibody/antigen binding will have taken place resulting in agglutination of the patient's red blood cells. The test tube is manually shaken to dislodge the centrifuged button of “clumped” cells at the bottom. A subjective determination is then made as to whether or not the dislodged cells are “clumped”, and to what extent.
During the mid-1900's, attempts were made to simplify this technique to minimize the subjective nature of the test and to reduce mistakes. It was recognized that a somewhat permanent record of the results of compatibility testing could be had by employing wettable, either non-absorbent, or in some cases absorbent, test slides or test cards having the requisite immunochemical reagents on at least a portion of their surfaces. U.S. Pat. Nos. 2,770,572, 2,850,430, 3,074,853, 3,272,319, 3,424,558, 3,502,437 and 3,666,421, and European Patent Application #0 104 881-A2 depict select examples of such test cards and related apparti.
It is standard bloodbanking practice to test for A, B and D (RHo) antigens on a sample of a person's blood (and to perform tests for other antigens in selected cases), and to crosscheck the results by testing the person's sera to determine the acquired antibodies that might be present. The former is referred to as “forward typing” or “direct test” while the latter is referred to as “reverse typing” or “indirect test”. The results from each of these typing exercises have to agree.
For the reverse or indirect test, for detecting antibodies in the serum or plasma of a patient, reagents containing blood cells having known antigens are mixed with a patient serum sample. The reactants are incubated for a period of time sufficient to permit agglutination of the red blood cells, which occurs when antibodies against those antigens are present. Such incubation typically ranges from about 10 minutes to about 40 minutes in modern testing. The mixture is then centrifuged, and if agglutinated blood cells are present, such agglutinates are clearly visible at the bottom of the reaction vessel, thus indicating the presence of antibodies in the sample directed against the known antigens on the red blood cells. If no antibodies are present in the sample directed against the known antigens on the red blood cells, agglutination does not occur, and this is indicated by the absence of agglutinated red cells after centrifugation.
Antibodies of the ABO blood grouping system are generally immunoglobulin M (IgM). These antibodies have ten antigen binding sites per molecule. The IgM antibody is large enough to span the distance between red blood cells, so that when they are centrifuged, the cells will be bound together in a lattice “cell-antibody-cell-antibody” and will remain clumped together in agglutinates. For example, if anti-A is added to blood group A or blood group AB cells and the mixture is centrifuged, the cells will remain in clumps when resuspended. With the same antibody, group O and group B cells will resuspend as individual cells. Agglutination facilitated by IgM antibodies is termed direct agglutination.
Anti-D antisera is now manufactured as monoclonal IgM or polyclonal IgG mixtures. These reagents can phenotype RBCs on immediate spin without the presence of high protein additives. The human IgG molecule was also chemically modified to unhinge the disulfide bond so it could have a larger span and act like a IgM molecule.
IgG antibodies cannot easily span the distance between cells which tend to repel each other in a saline environment. Thus, IgG will bind to red cell antigens matching its specificity, but will not directly agglutinate such red cells as effectively as the larger IgM antibodies will. The presence of IgG antibody bound to a red cell is thus usually detected by the addition of anti-IgG which will cause the requisite agglutination, resulting in a lattice of “red cell-IgG/Anti-IgG/IgG-red cell”.
Serum naturally contains IgG that will neutralize the anti-IgG antibody added to bind to red blood cells. Therefore, the serum must be removed before such anti-IgG is added to the cells. Tests for IgG bound to red cells in vivo are called direct antiglobulin tests. Tests for IgG bound to red cells in vitro are called indirect antiglobulin tests. Such antiglobulin tests are also called “Coombs” tests. This indirect antiglobulin test is a blood test used to determine whether there are IgG antibodies in a patient's serum to specified antigens on the surface of red blood cells. In the Coombs test, serum is incubated in the presence of reagent red cells to allow the antibodies to bind to antigens on the surface of the red cells. These IgG antibodies most often do not, by themselves, agglutinate the red cells, or only agglutinate them insufficiently to be detected visually by conventional techniques. Addition of a second antibody directed to human IgG is usually necessary to facilitate visible agglutination.
A convenient gel test and method of detecting antibodies or antigens is contemplated in this invention, wherein complexes of carrier-bound antibodies with antigens or carrier-bound antigens with antibodies in aqueous medium are made optically visible as before described. The carrier is preferably a gel or polymer and the antigens or antibodies as the case may be are bound to the carrier surface. The gel test contemplated herein is the Anti-Human Globulin Anti-IgG (Rabbit) MTS Anti-IgG Card™ for use with the ID-Micro Typing System™ (Micro Typing Systems, Inc., Pompano Beach, Fla., as disclosed in U.S. Pat. Nos. 5,338,689, 5,512,432, 5,863,802, and 6,114,179, the contents of which are incorporated herein by reference. Such a card may be used for both Direct and Indirect Antiglobulin Test. However, the invention is not limited to such test system and method but may be used with other formats besides gel such as for example, test tube, slide, solid phase and column agglutination technology (CAT) systems and methods, the latter whether in column or cassette form such as for instance the BioVue System™ of Ortho-Clinical Diagnostic Systems, Inc., Raritan, N.J., and might be used in any immunohematology system that employs incubation of the antibody and red cell antigen, regardless of test method.
In the ID-Micro Typing System™, the Direct test, which does not normally employ incubation, is accomplished by the employment of a gel card containing microtubes each of which contain an antibody incorporated into the gel matrix, and wherein diluted patient RBCs are placed on top of the gel carrier. Anti-human globulin (anti-IgG) is present in the gel. The card is centrifuged, which accelerates the reaction, if any, between the antibody reagent on the gel and the patient blood cells containing antigen, and also urges any cells toward the bottom of the microtubes. The gel in the microtubes act as a filter, however, and resist or impede downward movement of the particles in the microtube. As a result, the nature and distribution of the particles in the microtube after centrifuging provides a visual indication of whether any agglutination reaction occurred in the microtube, and if so, of the strength of that reaction.
If no agglutination reaction occurs, then all or virtually all of the red blood cells in the microtube pass downward during centrifuging, to the bottom of the microtube and form a pellet at the bottom. If there is a very strong reaction between the reagent and the red blood cells, virtually all of the red blood cells agglutinate, and large agglutinates form at the top of the microtube, above the gel contained therein; the gel or glass beads prevent the agglutinates from passing, during centrifuging, to the bottom of the column, so that after centrifuging the agglutinates remain on the surface of the gel.
If there is a reaction between the reagent and the blood cells, but this reaction is not as strong as the above described very strong reaction, then some but not all of the red blood cells agglutinate. The percentage of red blood cells that agglutinate and the size of the agglutinated particles both vary directly with the strength of the reaction. During centrifuging, the unreacted blood cells pass to the bottom of the column, and the distance that the agglutinated particles pass downward through the column depends on the size and number of those particles. Hence, the size of the pellet of red blood cells at the bottom of the microtube, and the extent to which the agglutinates penetrate into the gel in the microtube, are both inversely related to the strength of the reaction between the reagent and the red blood cells.
The instant invention is a method to reduce time to result in blood bank immunohematologic testing for tests that use incubation of the antibody and the red cell antigen. As discussed hereinabove, antigen-antibody reactions, including red cell typing reactions needing a incubation step, in immunohematology are detected by the visible agglutination of red blood cells or the evidence of hemolysis at the completion of testing. Such a test is for example, the Indirect or Reverse test as before mentioned, wherein patient antibodies are detected in sample plasma or serum by agglutination with diluted reagent RBCs. As stated, and in the ID-MTS System™ when for example conducting the Indirect test, patient sample plasma or serum is added to a microtube containing reagent RBCs that have been diluted in a low ionic strength solution (diluted MTS Diluent 2™), the card containing the microtube is incubated with agitation, followed by centrifugation and removal and reading of the card for agglutination. The instant invention allows for a decreased time for incubation due to potentiation of the antibody-antigen reaction as discussed below.
In the sensitization stage of hemagglutination, the antibody attaches to an antigen on the red blood cell. During this immunologic recognition stage antigenic determinants on the red blood cell combine with the antigen-binding site of the antibody molecule. The combination of an antigen and antibody is a random pairing of the two molecules determined largely by chance. Several factors influence the probability for this collision of antigen and antibody (Blaney K, Howard P: Basic and Applied Concepts of Immunohematology, St Louis, 2000, Mosby, and Vengelen-Tyler V: Technical manual, ed 12, Bethesda, Md, 1996, American Association of Blood Banks), and include the following:
Concentrations of Antibody and Antigen
The relative serum to cell ratio (i.e., the ratio of antigen on the red blood cell to antibody in the serum) will influence the probability of antigen-antibody combinations. Increasing the amount of serum in testing increases the concentration of antibodies available for binding to the red blood cell antigens. The number of antigen sites available on a per red blood cell basis also contributes to the strength of the antigen-antibody reaction.
Antigen Receptor Accessibility
The position of an antigen relative to the lipid bilayer of the red blood cell membrane contributes to its accessibility to antibody molecules, particularly IgG molecules. If the steric hindrance is decreased, the antibody molecules have a greater opportunity of interaction with the antigenic determinants.
Temperature of the Reaction Milieu
The temperature of the reaction influences the first stage of the agglutination reaction. In immunohematology most antibodies with clinical relevance are IgG immunoglobulins, which optimally react at temperatures of 37° C. In contrast, IgM antibodies are more reactive at lower temperatures, generally at or below room temperature. Providing the suitable temperature in the reaction enhances the sensitization step.
Incubation Time
Allowing adequate time for the combination of antigen and antibody to attain equilibrium is also a factor that enhances the first stage of the agglutination reaction.
PH
The optimal pH for hemagglutination is approximately pH 7.0. This pH is adequate for the majority of important red blood cell antibodies.
Ionic Strength
The lowering of the ionic strength of the test medium greatly enhances the rate at which antibodies bind to red blood cells. The use of low ionic strength solution as a potentiator of agglutination is a common practice in blood bank testing. For example, use of a buffer of about 0.03M is most useful. See Low and Messiter, Vox Sang 1974, Vol. 26, p. 53. Use of MTS Diluent 2™ (Micro Typing Systems, Inc., Pompano Beach, Fla.) is preferred.
All current commercial antibody detection test methodologies have the initial step of having the antigen (RBC) and the antibody (serum or plasma) incubated at 37° C. for a period of time, between 10-40 minutes and typically minutes. The instant invention provides for a significant reduction of time of incubation in an immunohematologic assay by employing continuous agitation and optionally, low ionic strength diluent. This reduction of test time can be realized no matter what specific test format is being used, whether test tube test, slide test, solid phase test system, microcolumn or microtube, or microplates, and regardless of matrix material, for instance, whether gel or glass bead is employed as matrix.
Blood bank testing has maximized the use of the factors previously outlined to produce diagnostic tests with appropriate sensitivity and specificity. The manipulation or combination of any of these variables of antigen-antibody reactions in test systems can reduce the time to result in blood bank testing. The instant invention is directed to reduction in incubation time required by use of continuous agitation while incubating. However, it will be understood that the actual amount of reduction in time can vary with the factors enumerated hereinabove, such as the ionic strength of the diluent, the presence or absence of enhancement agents (such as bovine albumin, polyethylene glycol, or proteolytic enzyme) the red blood cell/serum or plasma ratio, the initial temperature of the test sample and reagents, etc.
A review of the literature has not identified the previous use or disclosure of continuous agitation as a means to reduce the incubation time in immunohematology.